B77-25 Pirfenidone Analogs Induce the Integrated Stress Response via Gcn2-eif2α-atf4 to Inhibit Fibroblast Proliferation

Introduction

Pirfenidone exhibits pleiotropic antifibrotic and anti-inflammatory properties in experimental models and was the first ever antifibrotic medicine to reach patients in 2008. However, after more than 30 years of research, the drug’s mechanism of action (MOA) remains elusive because its poor potency precludes rigorous target identification. Until its MOA is understood, efforts to develop more effective drugs in this class will be difficult.

Methods

We establish structure-activity relationship assays (SAR) for the more potent pirfenidone analog GDC-3280 and its inactive analogs using quantitative live cell imaging and global proteomics. Based on robust SAR, we deploy a genome-wide CRISPR-knock-out screen in immortalized human foreskin fibroblasts.

Results

While GDC-3280 inhibits expression of several myofibroblast markers as previously reported, unbiased global proteomics reveal suppression of E2F targets, a G0/1 cell cycle arrest signature, and upregulation of ATF4 target genes. A CRISPR-KO screen identified EIF2AK4 (GCN2), a major stress-sensing kinase of the integrated stress response (ISR) pathway, as a key dependency for GDC-3280’s effect on fibroblast proliferation. Genetic deletion of GCN2 and co-treatment with an ISR inhibitor (ISRIB) alleviate the drug’s effect on fibroblast growth at concentrations near the clinically achievable range.

Conclusion

Our study suggests that Pirfenidone analog GDC-3280 may exert its therapeutic effect by inducing the integrated stress response through activation of GCN2, eIF2α and ATF4. Loss of EIF2AK4 (GCN2) leads to pulmonary veno-occlusive disease in humans, and, in animals, to worsening pulmonary fibrosis and pulmonary hypertension. GCN2 protein levels are reduced in the endothelium of diseased lungs with idiopathic pulmonary fibrosis and pulmonary hypertension, suggesting diseased lungs lack fully functional GCN2-mediated ISR activity. Therefore, restoration of the integrated stress response via activation of GCN2 may be of therapeutic benefit for fibrosing pulmonary disease.

This abstract is funded by: Anonymous donation to the Section of Molecular Medicine, Department of Radiation Oncology at UT Southwestern Medical Center